Description: The broad long-term objective of this research proposal is to understand the mechanisms by which the human immunodeficiency virus type 1 (HIV-1) regulates transcription of its genes. The HIV-1 Tat protein, acting through the TAR RNA element, activates the elongation efficiency of RNA polymerase II (RNAP II) complexes assembled on the HIV- 1 promoter. The laboratory has identified and purified an elongation factor, CAl50, from a fraction required for Tat activation in vitro (Sune et al., 1997). Overexpression of CA150 inhibits transcription from the HIV-1 promoter and the cellular a4 integrin promoter, but does not affect transcription from many other promoters (Sune and Garcia-Blanco, 1999). In vivo and in vitro experiments show that the inhibition is mediated at the level of elongation efficiency. CA15O associates with P-TEFb and with Tat-SF1, two other elongation factors known to play a role in HIV-1 transcription. During the next five years we propose to continue the exploration of the function of CAl50 and its role in HIV-1 transcript elongation; we propose to accomplish the following specific aims: 1. To determine the mechanism by which CA150 inhibits HIV-1 transcript elongation. The elements in the HIV-1 promoter that make it sensitive to CAl50 and the domains in CAl50 necessary for function will be identified. The mechanism of CA150 action will be probed in vivo and in vitro. 2. To characterize the factors that interact with CAl50 and to identify novel ones. The direct interaction of CAl50 with the C-terminal domain of RNAP II will be carefully dissected. Proteins that interact with the N-terminal domains of CAl50 will be identified and characterized. The association of CAl50 with other elongation factors (i.e., Tat-SF1 and P-TEFb) will be analyzed. 3. To analyze the properties and composition of elongation complexes assembled on the HIV-1 promoter. The function of CA150 and other elongation factors in the assembly and function of these complexes will be investigated. Better understanding of HIV-1 gene expression should enhance the possibility to develop novel therapies for AIDS and FIN-i infection.